Next-Generation Sequencing Panel Test in Myeloid Neoplasms and Evaluation with the Clinical Results

Abstract Objective: Myeloid malignancies are heterogeneous disorders due to defective hematopoiesis and myeloid differentiation of hematopoietic stem/progenitor cell. The molecular landscape of the diseases is complex. Molecular alterations are used for classification and evaluation of prognosis and treatment. We aimed to evaluate the advantages of the next-generation sequencing panel testing in myeloid malignancies and clinical outcomes. Materials and Methods: We evaluated the results of 54 patients who underwent next-generation sequencing myeloid panel testing, with fluorescent in situ hybridization (FISH), polymerase chain reaction results and the clinical outcomes. Target genes in the panel were ASXL1, CALR, CBL, CEBPA, CSF3R, DNMT3A, EZH2, FLT3, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SF3B1, SH2B3, SRSF2, TET2, TP53, U2AF1, and ZRSR2. Results: Diagnoses were acute myeloid leukemia, essential thrombocytosis, polistemia vera, primary myelofibrosis, hypereosinophilic syndrome (HES), chronic myeloid leukemia, myelodysplastic syndromes, chronic myelomonocytic leukemia. Twenty-eight missense, 8 frameshift, 5 stop gain, and 3 in-frame mutations were detected. A double mutation was detected in JAK-2 with next-generation sequencing in the patient who was given a false negative result due to polymerase chain reaction limitation. Conclusion: Screening multiple mutations simultaneously, is time and cost-effective. With the panel test, it is possible to determine the diagnosis, prognosis and targeted treatment options with a single test. Next-generation sequencing myeloid panel tests might be a powerful guide for clinicians.


Introduction
Myeloid malignancies consist of a heterogeneous group of disorders due to defective hematopoiesis of hematopoietic stem/progenitor cell and myeloid differentiation. Myeloproliferative neoplasms (MPN), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML) are the main disorders in these groups. 1 Genetic abnormalities are detected in 50-60% of the patients with acute myeloid leukemia by conventional cytogenetic and FISH method. 2 Cytogenetic abnormalities alone are not sufficient for AML formation, mutations such as FLT3 and RAS must be acquired additionally. Since cytogenetic abnormality was not detected in approximately half of AML cases, pathogenesis is explained by gene mutations. 3 Mutations classified as class 1 mutations; FLT3, KRAS, NRAS, CKİT, and JAK2 (signaling and kinase pathway), cause increased tyrosine kinase activity, triggering proliferation of the cell. mutations; class 2 mutations; CEBPA, NPM1 (transcription factors), and MLL, prevent apoptosis of the cell and disrupt its differentiation. 4 In addition, epigenetic mutations such as TET, ASXL1, IDH1, IDH2, EZH2, DNMT3, and RNA splicesome mutations such as U2AF1, SRSF2, ZRSR2, SF3B1, and tumor suppressors such as WT1 and TP53 have been blamed in the pathogenesis of AML. 5 Age, performance status of the patient, comorbid diseases, MDS, and myeloproliferative disease history as well as genetic characteristics are determinant in the prognosis of AML. In 2017, the genetic risk assessment of AML was updated by European Leukemia Network and classified as favorable, intermediate, and adverse groups. 6 Biallelic CEBPA mutation and NPM1 mutation without FLT3-ITD or with FLT3-ITD low are the genetic variations classified in the favorable group.
NPM1 mutation FLT3-ITD high and wild-type NPM1 without FLT3-ITD or with FLT3-ITD low are classified in the intermediate group. Wildtype NPM1 and FLT3-ITD high , RUNX1 mutation, ASXL1 mutation, and TP53 mutation are classified in the adverse group. 6 Screening of FLT3, NPM1, CEBPA, and KIT mutations is recommended also by National Comprehensive Cancer Network. 1 SF3B1 and actionable IDH1, IDH2 mutations are also advised to be tested. Common somatic mutations in AML are in FLT3, NPM1A, DNMT3A at a rate of 25-30% and IDH1/2, TET2 at a rate of 5-15%. Screening of these mutations is useful for diagnostic, prognostic, and treatment options. 1,7 Targeted therapy in recent years has led to an improvement in treatment. If a germline mutation is detected in the evaluation of the patient, treatment options can be changed. Individuals at risk in the family should be examined especially before stem cell transplantation. 7 Myelodysplastic syndrome is a clonal stem cell disease characterized by dysplasia and peripheral blood cytopenias. The main somatic mutations in MDS are TET2, ASXL1, SRSF2, RUNX1, TP53, EZH2, ZRSR2, SF3B1, and ETV6 mutations. 8 TP53 mutation is associated with short survival, high risk of AML transformation, increased blast percentage, and complex karyotype. 9 SF3B1, SRSF2, and U2AF1, splicesome group gene mutations, are seen in more than 50% of MDS patients. SF3B1 mutation is considered to be a good prognostic marker in MDS and is associated with the ring-sideroblast (RS) phenotype. SF3B1 mutation is seen in 60-90% of the patients with RS. 10 RAS mutation occurs in approximately 10-15% of cases and increases the risk of conversion to AML. FLT3 mutation is seen in 5% of the cases and is associated with a poor prognosis. TET2, ASXL1, and DNMT3A mutations are detected in 60-70% of MDS patients. ASXL1 mutation is known to be a poor prognostic marker. 11 Chronic myeloproliferative diseases were classified into essential thrombocytosis (ET), primary myelofibrosis (PM), chronic neutrophilic leukemia, chronic eosinophilic leukemia, chronic myeloid leukemia (CML), and unclassified myeloproliferative neoplasia by WHO in 2016. 12 JAK2, CALR, and MPL are driver mutations. JAK2 V617F mutation was detected in 95% of polistemia vera (PV) cases, 55% of ET cases, and 60% of PM cases. JAK2 exon 12 mutation is seen in approximately 3% of PV cases and these patients are younger and isolated erythrocytosis is more common. Disease outcomes are similar to those of JAK2 V617F-positive patients. MPL mutation is observed in 3% of ET cases and 7% of PM cases and in the presence of MPL mutations, older age, higher platelet count and erythropoietin levels, lower hemoglobin, and bone marrow cellularity are seen. CALR mutation is seen in 15-24% of ET and 25-35% of PM cases. TET2, EZH2, DNMT3A, ASXL1, CBL, IKZF1, IDH1/2, SH2B3, FLT3, SOCS, HMGA2, TP53, and NRAS/KRAS mutations are considered important for prognosis. 13,14 ASXL1 and EZH2 mutations are associated with poor prognosis in PM. 15 TET2 and IDH1/2 mutations facilitate leukemic transformation.
While so many mutations were identified in the myeloid malignancy diseases, it seems reasonable to screen them at once with the panel test. And also it is important to perform multigenic NGS panel tests at different times for clinical follow-up of the patients due to the risk of emergence of new somatic mutations.

Materials and Methods
We evaluated the results of our 54 patients who had myeloid panel testing due to myeloid malignancy in the last 1 year, retrospectively. This study was approved by Ataturk University Medical Faculty Clinical Research Ethics Committee (B.30.2.ATA.0.01.00/521). Written informed consent was obtained from all participants who participated in this study.
DNA quality assessment was done with the QIAxcel instrument. GeneRead Sequencing Q Kit was used to sequence the amplicon libraries on the GeneReader system. Variants were evaluated with Qiagen Clinical Insight Software which includes Clinvar, CADD, PolyPhen, SIFT, Mutation Taster, BLOSUM, PhyloP, MaxEntSan, Gene Splicer, B-SIFT, HGMD, COSMIC in silico tools.
In addition, FISH and polymerase chain reaction (PCR) test results and clinical history were evaluated.

Results
Myeloid panel results of the 58 patients evaluated; 3 were children, and 1 was diagnosed with biphenotypic acute leukemia hence they were excluded from the study. Six of our patients with AML died. No mutation was detected in one of these cases. Two other AML cases had FLT3, IDH1, EZH2, IDH2, NRAS, NPM1, DNMT3A, KRAS, and SF3B1 mutations. The sole MDS case with SRSF2, SETB1 mutations, and p53 deletion died.

Main Points
• Myeloid malignancies are heterogeneous diseases and the molecular landscape is complicated.
• Due to complex mutations and transformation of cells, screening of multiple genes is useful.
• Next-generation sequencing based technology allows time and cost effective results of multiple genes at once.
• Next-generation sequencing myeloid panel tests provide molecular biomarkers for diagnosis, prognosis, and targeted therapy.  19 and is in remission. IDH1/2 mutations are commonly seen with the co-existence of NPM1 and DNMT3A. Prognostic effect of IDH1/2 is conflicted. 20 The epigenetic basis of myeloid malignancies such as classification due to DNA methylation profile is a new issue. DNMT3A, ASLX, and TET2 mutations tend to occur in advanced ages and are associated with poor prognosis. Co-existence with other mutations could change clinical outcomes. Hypomethylating agents are therapy choices for these mutations but it is not approved yet. TP53 mutations are seen with complex genetic abnormalities. 1 In MPN patients, mutations other than driver mutations (JAK2, CALR, and MPL) provide information about prognosis. It was thought that the frequency of JAK2 mutations in our MPN patients was lower than the rates stated in the literature because the patients who requested a myeloid panel were atypical. A myeloid panel was not requested in classical patients who had already had JAK2 mutation with PCR. One of our PM patients had JAK2 V617F and ASLX1 mutations and the other had only JAK2 V617F mutation. Both of them were in a stable clinical state. It is reviewed that ASXL1, EZH2, IDH1, IDH2, TP53, or SRSF2 mutations are associated with a short survey and leukemic transformation and homozygote JAK2 V617F mutation causes more clinical symptoms and indicates poor prognosis in PM cases. 21  If we look at the limited aspects of our study, although the total number of patients we evaluated seems sufficient, our sample number is limited when divided into subgroups according to the diagnoses. With more sample numbers, clearer comparisons can be made between mutations and specific diagnoses.
In conclusion, evaluating more biomarkers in Informed Consent: Written informed consent was obtained from all participants who participated in this study.

Declaration of Interests:
The authors have no conflicts of interest to declare.
Funding: The authors declared that this study has received no financial support.